Diabetes and obesity (sometimes collectively referred to as “diabesity”) are interrelated in that obesity is known to exacerbate the pathology of diabetes and greater than 60% of diabetics are obese. Most human obesity is associated with insulin resistance and leptin resistance. In fact, it has been suggested that obesity may have an even greater impact on insulin action than diabetes itself (Sindelka et al., Physiol Res., 2002, 51, 85-91). Additionally, several compounds on the market for the treatment of diabetes are known to induce weight gain, a very undesirable side effect to the treatment of this disease.
Cardiovascular disease is also interrelated to obesity and diabetes. Cardiovascular disease encompasses a wide variety of etiologies and has an equally wide variety of causative agents and interrelated players. Many causative agents contribute to symptoms such as elevated plasma levels of cholesterol, including non-HDL cholesterol, as well as other lipid-related disorders. Such lipid-related disorders, generally referred to as dyslipidemia, include hyperlipidemia, hypercholesterolemia and hypertriglyceridemia among other indications. Elevated non-HDL cholesterol is associated with atherogenesis and its sequelae, including cardiovascular diseases such as arteriosclerosis, coronary artery disease, myocardial infarction, ischemic stroke, and other forms of heart disease. These rank as the most prevalent types of illnesses in industrialized countries. Indeed, an estimated 12 million people in the United States suffer with coronary artery disease and about 36 million require treatment for elevated cholesterol levels.
Epidemiological and experimental evidence has shown that high levels of circulating triglyceride (TG) can contribute to cardiovascular disease and a myriad of metabolic disorders (Valdivielso et al., 2009, Atherosclerosis. 207(2):573-8; Zhang et al., 2008, Circ Res. 1; 102(2):250-6). TG derived from either exogenous or endogenous sources is incorporated and secreted in chylomicrons from the intestine or in very low density lipoproteins (VLDL) from the liver. Once in circulation, TG is hydrolyzed by lipoprotein lipase (LpL) and the resulting free fatty acids can then be taken up by local tissues and used as an energy source. Due to the profound effect LpL has on plasma TG and metabolism in general, discovering and developing compounds that affect LpL activity are of great interest.
Metabolic syndrome is a combination of medical disorders that increase one's risk for cardiovascular disease and diabetes. The symptoms, including high blood pressure, high triglycerides, decreased HDL and obesity, tend to appear together in some individuals. It affects a large number of people in a clustered fashion. In some studies, the prevalence in the USA is calculated as being up to 25% of the population. Metabolic syndrome is known under various other names, such as (metabolic) syndrome X, insulin resistance syndrome, Reaven's syndrome or CHAOS. With the high prevalence of cardiovascular disorders and metabolic disorders there remains a need for improved approaches to treat these conditions
The angiopoietins are a family of secreted growth factors. Together with their respective endothelium-specific receptors, the angiopoietins play important roles in angiogenesis. One family member, angiopoietin-like 3 (also known as ANGPT5, ANGPTL3, or angiopoietin 5), is predominantly expressed in the liver, and is thought to play a role in regulating lipid metabolism (Kaplan et al., J. Lipid Res., 2003, 44, 136-143).
The human gene for angiopoietin-like 3 was identified and cloned as a result of searches of assembled EST databases. The full-length human cDNA codes for a polypeptide of 460 amino acids which has the characteristic structural features of angiopoietins: a signal peptide, an extended helical domain, a short linker peptide, and a globular fibrinogen homology domain (FHD). The mouse angiopoietin-like 3 cDNA was found to encode a 455 amino acid polypeptide with 76% identity to the human polypeptide. An alignment of angiopoietins showed that angiopoietin-like 3, unlike other family members, does not contain the motif of acidic residues determining a calcium binding site. Northern blot analysis revealed expression principally in the liver of adult tissues, with murine embryo Northern blots showing the presence of transcripts as early as day 15, suggesting that angiopoietin-like 3 is expressed early during liver development and that expression is maintained in adult liver. The mouse gene maps to chromosome 4, and the human gene was mapped to the 1p31 region (Conklin et al., Genomics, 1999, 62, 477-482).
KK obese mice have a multigenic syndrome of moderate obesity and a diabetic phenotype that resembles human hereditary type 2 diabetes. These mice show signs of hyperinsulinemia, hyperglycemia, and hyperlipidemia. A strain of KK mice called KK/San has significantly low plasma lipid levels despite signs of hyperinsulinemia and hyperglycemia. The mutant phenotype is inherited recessively, and the locus was named hypolipidemia (hypl). The locus maps to the middle of chromosome 4, and the gene was identified as angiopoietin-like 3 through positional cloning. Injection of recombinant adenoviruses containing the full-length mouse or human angiopoietin-like 3 cDNA in the mutant KK/San mice caused an increase in plasma levels of triglyceride, total cholesterol and non-esterified fatty acids (NEFA). Similarly, injection of recombinant angiopoietin-like 3 protein into the mutant mice increased levels of triglycerides and non-esterified fatty acids. (Koishi et al., Nat. Genet., 2002, 30, 151-157).
In another study focusing on the metabolic pathways of triglycerides in KK/San mice, overexpression of angiopoietin-like 3 resulted in a marked increase of triglyceride-enriched very low density lipoprotein (VLDL). Differences in the hepatic VLDL triglyceride secretion rate were not significant between wild-type KK and KK/San mice. However, studies with labeled VLDL suggested that the low plasma triglyceride levels in KK/San mice were primarily due to enhanced lipolysis of VLDL triglycerides rather than to enhanced whole particle uptake. The plasma apoB100 and apoB48 levels of KK/San mice were similar to wild-type KK mice. ApoCIII-deficient mice have a similar phenotype to KK/San mice, and ApoCIII is thought to modulate VLDL triglyceride metabolism through the inhibition of lipase-mediated hydrolysis of VLDL triglycerides. In vitro analysis of recombinant protein revealed that angiopoietin-like 3 directly inhibits lipoprotein lipase (LPL) activity (Shimizugawa et al., J. Biol. Chem., 2002, 277, 33742-33748).
Consistent with a role in lipid metabolism, angiopoietin-like 3 mRNA was found to be upregulated in C57BL/6J mice fed normal chow diets with 4% cholesterol and in mice treated with the liver X receptor (LXR) agonist T0901317. LXRs are ligand-activated transcription factors which play a role in the regulation of genes that govern cholesterol homeostasis in the liver and peripheral tissues. In addition to cholesterol metabolism, LXRs may also play a role in regulation of fatty acid metabolism. Treatment of HepG2 cells with natural or synthetic agents which activate LXR caused increased angiopoietin-like 3 expression. The promoter of the human angiopoietin-like 3 gene was found to contain an LXR response element. In addition, the promoter contained several potential binding sites for other transcription factors including HNF-1, HNF-4, and C/EBP. (Kaplan et al., J. Lipid Res., 2003, 44, 136-143).
Treatment of rodents with T0901317 is associated with triglyceride accumulation in the liver and plasma. The liver triglyceride accumulation has been explained by increased expression of the sterol regulatory element binding protein-1c (SREBP1c) and fatty acid synthase (FAS), both of which are targets of LXR. T0901317 failed to increase plasma triglyceride concentration in angiopoietin-like 3 deficient mice, while the stimulated accumulation of hepatic triglyceride was similar to that observed in treated wild type mice. The rise in plasma triglyceride in wild-type mice treated with T0901317 parallels an induction of angiopoietin-like 3 mRNA in the liver and an increase in plasma concentration of the protein. (Inaba et al., J. Biol. Chem., 2003, 278, 21344-21351).
Further studies addressed the mechanism of the increase in plasma free fatty acid (FFA) levels observed in KK/Snk mice treated with exogenous angiopoietin-like 3. Probe of fixed human tissues with a fluorescence-labeled angiopoietin-like 3 protein demonstrated strong binding only on adipose tissue. Furthermore, radiolabeled protein binding was examined in 3T3-L1 adipocytes and was found to be saturable and specific. Incubation of 3T3-L1 adipocytes with angiopoietin-like 3 led to enhanced release of FFA and glycerol into the culture medium. (Shimamura et al., Biochem. Biophys. Res. Commun., 2003, 301, 604-609).
In a study using streptozotocin-treated mice (STZ) to model the insulin-deficient state and db/db mice to model the insulin-resistant diabetic state, larger amounts of hepatic angiopoietin-like 3 were observed in diabetic mice as compared to control animals. Both models of diabetes showed hypertriglyceridemia, and the hyperlipidemia observed was explained at least partially by the increased expression of angiopoietin-like 3. These results suggested that angiopoietin-like 3 is a link between diabetes and dyslipidemia, with elevation promoting hyperlipidemia (Inukai et al., Biochem. Biophys. Res. Commun., 2004, 317, 1075-1079).
A subsequent study examined the regulation of angiopoietin-like 3 by leptin and insulin, both of which are key players in the metabolic syndrome. Angiopoietin-like 3 expression and plasma protein levels were increased in leptin-resistant db/db and leptin-deficient ob/ob mice relative to controls. Supplementation of ob/ob mice with leptin decreased angiopoietin-like 3 levels. The alterations in expression were associated with alterations in plasma triglyceride and free fatty acid levels. Gene expression and plasma protein levels were also increased in insulin-deficient STZ-treated mice. (Shimamura et al., Biochem Biophys Res Commun, 2004, 322, 1080-1085).
In accord with its membership in the angiopoietin family, recombinant angiopoietin-like 3 protein was found to bind to αvβ3 integrin and induced integrin αvβ3-dependent haptotactic endothelial cell adhesion and migration. It also stimulated signal transduction pathways characteristic for integrin activation. Angiopoietin-like 3 strongly induced angiogenesis in the rat corneal angiogenesis assay. (Camenisch et al., J. Biol. Chem., 2002, 277, 17281-17290).
Genome-wide association scans (GWAS) surveying the genome for common variants associated with plasma concentrations of HDL, LDL and triglyceride were undertaken by several groups. The GWAS studies found an association between triglycerides and single-nucleotide polymorphisms (SNPs) near ANGPTL3 (Willer et al., Nature Genetics, 2008, 40(2):161-169).
U.S. Pat. No. 7,267,819, application U.S. Ser. No. 12/128,545, and application U.S. Ser. No. 12/001,012 generally describe angiopoietin-like 3 agonists and antagonists.
PCT publications WO/02101039 (EP02733390) and WO/0142499 (U.S. Ser. No. 10/164,030) disclose a nucleic acid sequence complementary to mouse angiopoietin-like 3 (Ryuta, 2002; Ryuta, 2001).
There is a currently a lack of acceptable options for treating cardiovascular and metabolic disorders. It is therefore an object herein to provide compounds and methods for the treatment of such diseases and disorder.
The potential role of angiopoietin-like 3 in lipid metabolism makes it an attractive target for investigation. Antisense technology is emerging as an effective means for reducing the expression of certain gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of angiopoietin-like 3.